Nucleic Acids Research, Vol 24, Issue 24 5026-5033, Copyright © 1996 by Oxford University Press
EM Golenberg, A Bickel and P Weihs
We characterized the behavior of polymerase chain reactions (PCR) using
degraded DNA as a template. We first demonstrated that fragments larger
than the initial template fragments can be amplified if overlapping
fragments are allowed to anneal and extend prior to routine PCR.
Amplification products increase when degraded genomic DNA is pretreated by
polymerization in the absence of specific primers. Secondly, we measured
nucleotide uptake as a function of template DNA degradation. dNTP
incorporation initially increases with increasing DNA fragmentation and
then declines when the DNA becomes highly degraded. We demonstrated that
dNTP uptake continues for >10 polymerization cycles and is affected by
the quality and quantity of template DNA and by the amount of substrate
dNTP. These results suggest that although reconstruction of degraded DNA
may allow amplification of large fragments, reconstructive polymerization
and amplification polymerization may compete. This was confirmed in PCR
where the addition of degraded DNA reduced the resultant product. Because
terminal deoxynucleotidyl transferase activity of Taq polymerase may
inhibit 3' annealing and restrict the length of template reconstruction, we
suggest modified PCR techniques which separate reconstructive and
amplification polymerization reactions.
ARTICLES
Effect of highly fragmented DNA on PCR
Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA. egolenb@biology.biosci.wayne.edu
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